Nucleic acid hybridizations are commonly used in genetic research, biochemical research and clinical diagnostics. In basic nucleic acid hybridization assays, single stranded nucleic acid (either DNA or RNA) is hybridized to a labeled nucleic acid probe and the resulting duplex is detected.
Diagnostic assays based on hybridization frequently require amplification of the nucleic acid in the sample under test. Usually a polymerase chain reaction (PCR) is used for such amplification. However, many clinical laboratories and most doctors' offices are resistant to PCR because of its complexity and the possibility of amplifying contaminants. Assays which do not require amplification are more popular because, generally speaking, they are less labor intensive and take a shorter period of time to perform. There is, however, a growing need for non-amplified assays which have improved sensitivity and accuracy.
There are essentially three methods for improving sensitivity in non-amplified DNA probe assays: (1) primer extension, (2) extended incubation time and (3) multi-label macromolecular complexes. Some prior efforts to provide assays which do not require amplification are those shown in U.S. Pat. Nos. 5,175,270, 5,124,246, 4,925,785 and 4,372,745.
U.S. Pat. No. 5,175,270 discloses the use of multi-layered DNA constructs in the context of nucleic acid hybridization assays to reduce background noise and to initially enhance signal strength for very low copy sequences at short exposure times (overnight v. 2-6 weeks). The outermost layer of a given DNA matrix has single stranded sequences exposed to the surface which hybridize with a predetermined nucleic acid sequence. The assay formats specifically mentioned are Southern blotting hybridizations or dot hybridizations although others also are envisioned.
U.S. Pat. No. 5,124,246 discloses a scheme for binding multiple labelled probes onto a given target DNA employing branched DNA in a multi-layered oligo probe complex. This provides multiple binding sites for a specific set of single label oligo probes.
U.S. Pat. No. 4,925,785 discloses, in the context of a nucleic acid hybridization assay, employing multiple fluorescent labels on very large random coiled polymer support material to which oligonucleotide probes are attached to amplify signal. Covalent linkages are used to attach the labels and the probes to the polymer. Hetero- and homogeneous assay formats are disclosed.
U.S. Pat. No. 4,372,745 discloses the use of encapsulated florescent materials as part of a signal enhancing system in the context of immunologically based assays. The multiple labels are released as part of the detection step. A variety of assay formats are taught.
Assays based on ECL are well known in the art and are finding expanding applications because of their accuracy, ease of use and freedom from radioactive materials.
A particularly useful ECL system is described in a paper by Yang et al, Bio/Technology, 12:193-194 (February 1994). See also a paper by Massey, Biomedical Products, October 1992 as well as U.S. Pat. Nos. 5,235,808 and 5,310,687, the contents of these papers and patents being incorporated herein by reference.
ECL processes have been demonstrated for many different molecules by several different mechanisms. In Blackburn et al (1991) Clin. Chem. Vol. 37, No. 9, pp. 1534-1539, the authors used the ECL reaction of ruthenium (II) tris(bipyridyl), Ru(bpy).sub.3.sup.2+, with tripropylamine (TPA) (Leland et al (1990) J. Electrochem. Soc. Vol. 137, pp. 3127-31) to demonstrate the technique. Salts of Ru(bpy).sub.3.sup.2+ are very stable, water-soluble compounds that can be chemically modified with reactive groups on one of the bipyridyl ligands to form activated species with which proteins, haptens, and nucleic acids are readily labeled. The activated form of the Ru(bpy).sub.3.sup.2+ used by Blackburn et al was Ru(bpy).sub.3.sup.2+ -NHS ester: ##STR1##
FIG. 1 shows the proposed mechanism for the ECL reaction of Ru(bpy).sub.3.sup.2+ and TPA. Ru(bpy).sub.3.sup.2+, the label, is oxidized at the surface of an electrode, forming the strong oxidant, Ru(bpy).sub.3.sup.3+. Simultaneously, TPA which is present in large molar excess in the solution, is oxidized at the electrode to form the cation radical TPA.sup.+.multidot., which rapidly and spontaneously loses a proton to form the radical TPA. Ru(bpy).sub.3.sup.3+, a strong oxidant and TPA, a strong reductant, react to form the excited state of the ruthenium complex, Ru(bpy).sub.3.sup.2+*, as well as other inactive products. The energy necessary for formation of the excited state arises from the large difference in electrochemical potentials of the Ru(bpy).sub.3.sup.3+ and the TPA. The excited-state Ru(bpy).sub.3.sup.2+* decays through a normal fluorescence mechanism, emitting a photon at 620 nm. This process regenerates the original form of the Ru(bpy).sub.3.sup.2+, which is free to cycle multiple times through the reaction sequence. Each ECL-active label, therefore, can emit many photons during each measurement cycle, thereby enhancing the detection of the label.
Quantification of the Ru(bpy).sub.3.sup.2+ label can be readily automated with relatively uncomplicated instrumentation. FIG. 2 is a diagram of the essential components of instruments under development for automated immunoassays and DNA probe assays. The heart of the instrument is the electrochemical flow-cell, containing the working electrodes and counter electrodes for initiation of the ECL reaction. Both of the electrodes are fabricated from gold, but other materials have been used with various degrees of success. A potentiostat (not shown) applies various voltage waveforms to the electrodes, and a single photomultiplier tube (PMT) detects the light emitted during the ECL reaction. An Ag/AgCl reference electrode is placed in the fluid path downstream from the flow cell, and a peristaltic pump is used to draw various fluids through the flow cell. In a typical sequence, the assay fluid is drawn from a test tube into the flow cell and the label is quantified by applying a ramp voltage to the electrodes and measuring the emitted light. After the measurement, a high-pH cleaning solution is drawn into the cell for an electrochemical cleaning procedure. A conditioning solution is then drawn into the cell, and a voltage waveform is applied that leaves the surfaces of the electrodes in a highly reproducible state, ready for the next measurement cycle.
The ECL reaction can be efficiently initiated by many different voltage waveforms. FIG. 3 illustrates measurements of the working electrode current and the ECL intensity induced by the application of a triangle wave to the electrodes. The applied voltage as shown is actually the voltage measured at the Ag/AgCl reference electrode and includes the effects of a significant uncompensated resistance; consequently, the actual voltage applied at the working electrode is substantially less than that depicted. The triangle waveform rises from 565 to 2800 mV at a rate of 750 mV/s and then decreases at the same rate to 1000 mV. The current that flows in the cell is primarily the result of the oxidation of the TPA and the hydrolysis of water. Oxidation of both the TPA and Ru(bpy).sub.3.sup.2+ becomes evident when the applied voltage reaches .about.1100 mV and produces a luminescence. The intensity of the luminescence increases with the applied voltage until the TPA at the surface of the electrode is depleted, resulting in decreased intensity. The intensity of the observed luminescence is great enough that it can easily be measured with conventional PMTs operating either in photon-counting or current modes.
The system also includes magnetic beads suitably coated to capture the molecules of interest and bring them to the electrode surface. For this purpose, a magnet is positioned under the electrode to bring the magnetic beads coated with the target molecules.
In a conventional immunoassay using the ECL system, anti-target antibodies may be bound to the magnetic beads. Additionally, anti-target antibodies which recognize a different epitope or part of the target are combined with an ECL label to form what may be termed "reporter" or "detector" molecules. By incubating the target molecule with the magnetic beads and the labeled anti-target antibodies, a "sandwich" is formed by virtue of the attachment of the two separate antibodies to the target (antigen) at different sites. This sandwich is then drawn into the flow cell and mixed with buffer solution containing a precursor. An applied magnetic force captures the magnetic beads on the electrode surface thus stabilizing the target molecule and its attached labeled reporter for maximum detection by PMT. Unbound reagents from the sample mix are washed away by continued flow of buffer solution.
After the sample is captured, the ECL measurement is performed by application of electrical potential to the working electrode. This gives a clean signal-to-noise ratio in that only those labels which are bound to the "sandwich" that are surrounded by the precursor and in contact with the electrode emit light. Relatively little interference results from background presented by the buffer or otherwise.
The ECL system may also be used for nucleic acid hybridization-based assays with oligonucleotides being used for hybridization in lieu of the antibodies referred to above. However, for this purpose, the conventional practice has been to amplify the nucleic acids by using polymerase chain reaction (PCR) or nucleic-acid sequence-based amplification (NASBA). While the ECL process, using amplification is effective, there is a need to be able to effectively carry out the ECL assay without amplification for reasons noted earlier. The sensitivity of non-amplified nucleic acid assays to date has been between 10.sup.3 -10.sup.6 copies of DNA (data not shown). Accordingly, the principal object of the present invention is to provide such an ECL assay. other objects will also be apparent from the description of the invention which follows.